Field of the Invention
The present invention relates to nanoparticles, having an optional plasmonics-active shell and functionalized with at least one photoactivatable pharmaceutical agent, that are capable of converting energy from one energy level to an energy level capable of activating the photoactivatable pharmaceutical agent, methods for the production of the nanoparticles and methods for their use, particularly in the treatment of cell proliferation disorders.
Description of the Related Art
More than a million individuals are diagnosed with cancer in the United States each year, and annual cancer diagnoses worldwide number above ten millon.A Although traditional approaches such as surgery, chemotherapy, radiation therapy, photodynamic therapy (PDT), and psoralen+UVA (PUVA) have shown some success in treating certain types of cancer, there is a strong need to develop effective, universally applicable, and non-invasive means of therapy. To that end, the rapidly developing field of nanomedicine has attempted to leverage the untapped potential of nanomaterials as a means of improving drug targeting to and uptake by tumors, locally activating therapeutic agents, and limiting side-effects which may negatively impact patients' quality of life.B 
These attempts to use nanomaterials to cure cancer have typically taken one of three approaches. In the first approach, nanoparticles have been used to aid in transport and delivery of chemotherapeutic agents.C Such methodologies have shown some potential, particularly in reducing the unpleasant side-effects associated with chemotherapy.D In a second approach, the nanoparticles themselves have been used as a means of enhancing the normal effects of some more traditional treatment techniques.E Two promising techniques which fall into this category are induction of hyperthermia by illuminating gold nanoshells with infrared light,F or enhanced reactive oxygen species (ROS) generation using solid gold nanoshells and X-ray radiation.G The third use of nanomaterials for cancer therapy is combination of ROS-generating PDT drugs with nanomaterials which emit visible light when excited by X-ray radiation (scintillators).H 
Of these three approaches, the third is perhaps the most intriguing and exhibits some unique advantages. In theory, only those cells which both take up the nanodrug and receive X-ray radiation stand a significant chance of dying during treatment. As a result, the side-effects associated with non-specific uptake of chemotherapeutic drugs should be greatly reduced. At the same time, radiation doses should theoretically be able to be reduced to a point where systemic effects are minor or nonexistent. Unfortunately, while such an approach with traditional PDT drugs may have potential in well-oxygenated tissue, reduced ROS generation in the inherently hypoxic environment of many tumorsI is likely to limit the broader utility of ROS-dependent X-ray activatable therapies.
The field of nanobiotechnology has experienced an explosive growth due to improved understanding of the characteristics and properties of nanoparticles and to rapid advances in the methods for their fabrication. With ongoing improvements in the techniques and technology needed for consistent production of nanomaterials, as well as a continually-improving understanding of their characteristics and potential, there has been a steady increase in the variety of laboratory-fabricated nanoparticles. Even with these improvements in the research laboratory, however, the types of particles which are commercially available are still limited to just a few categories.
Two of the first types of nanoparticles reliably synthesized in the laboratory were solid metal nanospheres of gold and silver,1 typically produced in aqueous solution using a metal salt and a suitable reductant such as sodium citrate or sodium borohydride. Improvements in wet synthetic techniques since these early studies have expanded the range of solid noble metal nanoparticle shapes to include silver rods,2-5 plates,6-10 prisms11 and cubes,12,13 as well as gold rods,14-24 disks,25-27 plates,28-30 prisms,31-33 cubes34,35 and stars.36-38 Each of these size-tunable particle shapes and morphologies exhibit unique plasmonic properties which can strongly enhance electromagnetic fields, making them useful for both intracellular and extracellular biochemical sensing of biotargets such as DNA and mRNA,39-48 or proteins and peptides49,50 via surface plasmon resonance39,51-54 or surface-enhanced Raman scattering (SERS).40-48,55 An added advantage of noble metal nanoparticles (e.g., gold and silver) is that they are easily functionalized and essentially non-toxic to cells and higher organisms, making in vivo and in vitro bioanalyses practical.
Somewhat more recently, dye-doped56-63 and dye-bound64 silicon dioxide (SiO2) nanoparticles have also found significant use as substrates for biochemical sensing when functionalized with nucleic acids, peptides or proteins.65 and refs therein Like gold and silver, SiO2 is particularly appealing for in vivo and in vitro sensing because it is easily fabricated, easily functionalized and relatively non-toxic.
Intrinsically fluorescent semiconductor quantum dots (e.g. Cd:Te, Cd:Se, Zn:S, etc.) have been used for a range of applications including analytical chemistry, biochemical sensing, and study of cellular uptake, fate and transport.66-69 The inherent toxicity of cadmium limits the utility of these materials in vivo, but they are eminently suited to a wide range of short-term in vitro studies during which longer-term toxicity is not an issue.
One approach to minimize the toxicity discussed above is to coat intrinsically-toxic nanomaterials with SiO2,70-75 thereby limiting the bioavailability of whatever toxins are present. This approach, in fact, has the potential to passivate a wide range of nanomaterials or, alternatively, allow more facile surface functionalization with biochemically-sensitive species. To this end, a number of researchers have added SiO2 shells to quantum dots,70-75 iron oxide,76-78 or solid noble metal nanoparticles.79-84 Alternatively, SiO2 nanoparticles themselves can be coated with gold or silver to produce plasmonic nanoshells which can be tuned from the near UV to the near IR.85-95 Various photochemical methods have been reported for the fabrication of gold nanoparticles and gold films [A. Pal, T. Pal, D. L. Stokes, and T. Vo-Dinh, “Photochemically prepared gold nanoparticles: A substrate for surface-enhanced Raman scattering”, Current Science, 84, 1342-1346 (2003); M. Volcan, D. L. Stokes and T. Vo-Dinh “A Sol-Gel Derived AgCl Photochromic Coating on Glass for SERS Chemical Sensor Application”, Sensors and Actuators B, 106, 660-667 (2005)] A. Pal, D. L. Stokes and T. Vo-Dinh, “Photochemically Prepared Gold Metal film in a Carbohydrate-based Polymer: a Practical Solid substrate for Surface-enhanced Raman Scattering, Current Science, 87, 486-491 (2004) and references therein].
There are also wet chemistry methods described in the literature [Oldenburg, S. J., Averitt, R. D., Westcott, S. L., and Halas, N. J. Nanoengineering of Optical Resonances. Chemical Physics Letters 288, 243-247 (1998); Jensen, R. A, Sherin, J. and Emory, S. R. Single Nanoparticle Based Optical pH Probe. Applied spectroscopy, 61, 8, 832-838 (2007); Oldenburg S. J., Westcott S. L., Averitt R. D., and Halas, N. J. Surface enhanced Raman scattering in the near infrared using metal nanoshell substrates. Journal of Chemical Physics, 111, 10, 4729-4735 (1999); and refrences therein]. An approach to form a gold shell around a core material is seed-mediated growth. The first step involves the use of chemical linkers to attach small Au or Ag seeds (from few nanometers to larger seeds depending on the core dimension) on the core surface. Several linkers can be used; a direct approach commonly described in the literature is to aminate the surface of the material to allow the adsorption of the seeds. Molecules with dual functionality act as linkers; an amino group to adsorb to the seeds or a thiol group to covalently bond the seeds on one side, and a carboxy-group, phosphonate-group, sulfonate-group on the other side to bind to the core surface, such as Y2O3, silica, polystyrene. Other approaches include using different coupling chemistry to bind two chemical groups attach to the core surface and the seeds. EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) -mediated coupling chemistry, for instance, enables the crosslinking of an amine and a carboxyl group. N-hydroxysuccinimide (NHS) coupling chemistry enables the formation of an amide bond between a primary amine and the succinimide ester. The second step in the seed mediated growth involves the attachment of additional gold, silver, silica, titania, alumina, yttria, etc. onto the nucleated seeds. As an example, a common approach to ripening of a gold layer is to use potassium carbonate and HAuCl4 in the presence of formaldehyde. Alkoxides are frequently employed for thickening of silica or titania shells, and similar precursor compounds can be used when forming shells of alumina, yttria, etc.
Phototherapy There are two main types of reactions in phototherapy:
Type I reactions involve electrons and hydrogen atoms, which are transferred between photo-active molecules (also called photosensitizers) and substrates or solvent molecules. Oxygen may participate in subsequent reactions: e.g., psoralens in photopheresis and PUVA.
Type II reactions involve singlet oxygen formation by energy transfer from PA molecules in the lowest triplet state to oxygen in the ground state: e.g., photodynamic therapy (PDT).
Psoralen System. Psoralen (FIG. 1) is the parent compound in a family of natural products known as furocoumarins. Psoralens are a class of photo-mutagenic and photo-chemotherapeutic molecules that covalently modify nucleic acids. They belong the family of small molecules that intercalate into and photoalkylate double stranded DNA. The primary target of psoralens are thymidine residues, and these molecules form both monoadducts and interstrand crosslinks The reaction takes place between the 3,4 (pyrone) or 4′,5′ (furan) double bonds of the psoralen and the 5,6 double bond in pyrimidines.
Psoralen compounds absorb UVA and UVB photons, and emit visible light. FIGS. 2A and 2B show excitation and emission fluorescence spectra of psoralens. [Source: T. Vo-Dinh et al, J. Agric. Food Chem., 36, 335 (1988)]
U.S. Pat. No. 6,235,508 further teaches that psoralens are naturally occurring compounds which have been used therapeutically for millennia in Asia and Africa. The action of psoralens and light has been used to treat vitiligo and psoriasis (PUVA therapy; Psoralen Ultra Violet A). Psoralen is capable of binding to nucleic acid double helices by intercalation between base pairs; adenine, guanine, cytosine and thymine (DNA) or uracil (RNA). Upon sequential absorption of two UV-A photons, psoralen in its excited state reacts with a thymine or uracil double bond and covalently attaches to both strands of a nucleic acid helix. The crosslinking reaction appears to be specific for a thymine (DNA) or a uracil (RNA) base. Binding proceeds only if psoralen is intercalated in a site containing thymine or uracil, but an initial photoadduct must absorb a second UVA photon to react with a second thymine or uracil on the opposing strand of the double helix in order to crosslink each of the two strands of the double helix, as shown below. This is a sequential absorption of two single photons as shown, as opposed to simultaneous absorption of two or more photons.

In addition, the reference teaches that 8-MOP is unsuitable for use as an antiviral, because it damages both cells and viruses. Lethal damage to a cell or virus occurs when the psoralen is intercalated into a nucleic acid duplex in sites containing two thymines (or uracils) on opposing strands but only when it sequentially absorbs 2 UVA photons and thymines (or uracils) are present. U.S. Pat. No. 4,748,120 of Wiesehan is an example of the use of certain substituted psoralens by a photochemical decontamination process for the treatment of blood or blood products.
Additives, such as antioxidants are sometimes used with psoralens, such as 8-MOP, AMT and I-IMT, to scavenge singlet oxygen and other highly reactive oxygen species formed during photoactivation of the psoralens. It is well known that UV activation creates such reactive oxygen species, which are capable of seriously damaging otherwise healthy cells. Much of the viral deactivation may be the result of these reactive oxygen species rather than any effect of photoactivation of psoralens. Regardless, it is believed that no auto vaccine effect has been observed.
Research in this field over-simplifies mechanisms involved in the photoactivating mechanism and formation of highly reactive oxygen species, such as singlet oxygen. Both may lead to inactivating damage of tumor cells, viruses and healthy cells. However, neither, alone or combined, lead to an auto vaccine effect. This requires an activation of the body's own immune system to identify a malignant cell or virus as threat and to create an immune response capable of lasting cytotoxic effects directed to that threat. It is believed, without being limiting in any way, that photoactivation and the resulting apoptosis of malignant cells that occurs in extracorporeal photophoresis causes the activation of an immune response with cytotoxic effects on untreated malignant cells. While the complexity of the immune response and cytotoxic effects is fully appreciated by researchers, a therapy that harnesses the system to successfully stimulate an auto vaccine effect against a targeted, malignant cell has been elusive, except for extracorporeal photopheresis for treating lymphoma.
Midden (W. R. Midden, Psoralen DNA photobiology, Vol I1 (ed. F. P. Gaspalloco) CRC press, pp. 1. (1988) has presented evidence that psoralens photoreact with unsaturated lipids and photoreact with molecular oxygen to produce active oxygen species such as superoxide and singlet oxygen that cause lethal damage to membranes.
U.S. Pat. No. 6,235,508 teaches that 8-MOP and AMT are unacceptable photosensitizers, because each indiscriminately damages both cells and viruses. Studies of the effects of cationic side chains on furocoumarins as photosensitizers are reviewed in Psoralen DNA Photobiology, Vol. I, ed. F. Gaspano, CRC Press, Inc., Boca Raton, Fla., Chapter 2. U.S.
U.S. Pat. No. 6,235,508 gleans the following from this review: most of the amino compounds had a much lower ability to both bind and form crosslinks to DNA compared to 8-MOP, suggesting that the primary amino functionality is the preferred ionic species for both photobinding and crosslinking.
U.S. Pat. No. 5,216,176 of Heindel discloses a large number of psoralens and coumarins that have some effectiveness as photoactivated inhibitors of epidermal growth factor. Halogens and amines are included among the vast functionalities that could be included in the psoralen/coumarin backbone. This reference is incorporated herein by reference.
U.S. Pat. No. 5,984,887 discloses using extracorporeal photopheresis with 8-MOP to treat blood infected with CMV. The treated cells as well as killed and/or attenuated virus, peptides, native subunits of the virus itself (which are released upon cell break-up and/or shed into the blood) and/or pathogenic noninfectious viruses are then used to generate an immune response against the virus, which was not present prior to the treatment.
A survey of known treatment methods reveals that these methods tend to face a primary difficulty of differentiating between normal cells and target cells when delivering treatment, often due to the production of singlet oxygen which is known to be non-selective in its attack of cells, as well as the need to perform the processes ex vivo, or through highly invasive procedures, such as surgical procedures in order to reach tissues more than a few centimeters deep within the subject.
U.S. Pat. No. 5,829,448 describes sequential and simultaneous two photon excitation of photo-agents using irradiation with low energy photons such as infrared or near infrared light (NRI). A single photon and simultaneous two photon excitation is compared for psoralen derivatives, wherein cells are treated with the photo agent and are irradiated with NRI or UV radiation. The patent suggests that treating with a low energy irradiation is advantageous because it is absorbed and scattered to a lesser extent than UV radiation. However, the use of NRI or UV radiation is known to penetrate tissue to only a depth of a few centimeters. Thus any treatment deep within the subject would necessarily require the use of ex vivo methods or highly invasive techniques to allow the irradiation source to reach the tissue of interest. Also, this patent does not describe initiation energy sources emitting energy other than UV, visible, and near infrared energy; energy upgrading other than within the range corresponding to UV and IR light, and downgrading from high to low energy.
Chen et al., J. Nanosci. and Nanotech., 6:1159-1166 (2006); Kim et al., JACS, 129:2669-2675 (2007); U.S. 2002/0127224; and U.S. Pat. No. 4,979,935 each describe methods for treatment using various types of energy activation of agents within a subject. However, each suffers from the drawback that the treatment is dependent on the production of singlet oxygen to produce the desired effect on the tissue being treated, and is thus largely indiscriminate in affecting both healthy cells and the diseased tissue desired to be treated.
U.S. Pat. No. 6,908,591 discloses methods for sterilizing tissue with irradiation to reduce the level of one or more active biological contaminants or pathogens, such as viruses, bacteria, yeasts, molds, fungi, spores, prions or similar agents responsible, alone or in combination, for transmissible spongiform encephalopathies and/or single or multicellular parasites, such that the tissue may subsequently be used in transplantation to replace diseased and/or otherwise defective tissue in an animal. The method may include the use of a sensitizer such as psoralen, a psoralen-derivative or other photosensitizer in order to improve the effectiveness of the irradiation or to reduce the exposure necessary to sterilize the tissue. However, the method is not suitable for treating a patient and does not teach any mechanisms for stimulating the photo sensitizers, indirectly.
U.S. Pat. No. 5,957,960 discloses a two-photon excitation device for administering a photodynamic therapy to a treatment site within a patient's body using light having an infrared or near infrared waveband. However, the reference fails to disclose any mechanism of photoactivation using energy modulation agent that converts the initiation energy to an energy that activates the activatable pharmaceutical agent and also use of other energy wavebands, e.g., X-rays, gamma-rays, electron beam, microwaves or radio waves.
U.S. Pat. No. 6,235,508 discloses antiviral applications for psoralens and other photoactivatable molecules. It teaches a method for inactivating viral and bacterial contaminants from a biological solution. The method includes mixing blood with a photosensitizer and a blocking agent and irradiating the mixture to stimulate the photo sensitizer, inactivating substantially all of the contaminants in the blood, without destroying the red blood cells. The blocking agent prevents or reduces deleterious side reactions of the photosensitizer, which would occur if not in the presence of the blocking agent. The mode of action of the blocking agent is not predominantly in the quenching of any reactive oxygen species, according to the reference.
Also, U.S. Pat. No. 6,235,508 suggests that halogenated photosensitizers and blocking agents might be suitable for replacing 8-methoxypsoralen (8-MOP) in photopheresis and in treatment of certain proliferative cancers, especially solid localized tumors accessible via a fiber optic light device or superficial skin cancers. However, the reference fails to address any specific molecules for use in treating lymphomas or any other cancer. Instead, the reference suggests a process of photopheresis for antiviral treatments of raw blood and plasma.
U.S. Pat. No. 6,235,508 teaches away from 8-MOP and 4′-aminomethyl-4,5′,8-trimethylpsoralen (AMT) and many other photoactivatable molecules, which are taught to have certain disadvantages. Fluorescing photosensitizers are said to be preferred, but the reference does not teach how to select a system of fluorescent stimulation or photoactivation using fluorescent photosensitizers. Instead, the fluorescing photosensitizer is limited to the intercalator that is binding to the DNA. The reference suggests that fluorescence indicates that such an intercalator is less likely to stimulate oxygen radicals.
U.S. published application 2002/0127224 discloses a method for a photodynamic therapy comprising administering light-emitting nanoparticles and a photoactivatable agent, which may be activated by the light re-emitted from the nanoparticles via a two-photon activation event. An initiation energy source is usually a light emitting diode, laser, incandescent lamp, or halogen light, which emits light having a wavelength ranging from 350 to 1100 nm. The initiation energy is absorbed by the nanoparticles. The nanopartuicles, in turn, re-emit light having a wavelength from 500 to 1100 nm, preferably, UV-A light, wherein the re-emitted energy activates the photoactivatable agent. Kim et al., (JACS, 129:2669-75, Feb. 9, 2007) discloses indirect excitation of a photosensitizing unit (energy acceptor) through fluorescence resonance energy transfer (FRET) from the two-photon absorbing dye unit (energy donor) within an energy range corresponding to 300-850 nm. These references do not describe initiation energy sources emitting energy other than UV, visible, and near infrared energy; energy upgrading other than within the range corresponding to wavelength of 350-1100 nm, and downgrading from high to low energy.
These references fail to disclose any mechanism of photoactivation of photoactivatable molecules other than by direct photoactivation by UV, visible, and near infrared energy.
Therefore, there still exists a need for better and more effective treatments that can more precisely target the diseased cells without causing substantial side-effects or collateral damages to healthy tissues, and which are capable of treating disorders by non-invasive or minimally invasive techniques.
Cell Penetrating Peptides (CPP) and Nuclear Targeting Peptides (NTP) for Cellular Delivery of Nanoparticles
An important element involves effective intracellular delivery of the nanoparticle-based drug systems into the cells and inside the nucleus in order to bind to DNA. Viral vectors have been proposed for DNA delivery but these approaches are limited by non-specificity and inherent risks of virus-induced complications. Liposomes and micelles have been used for the delivery of water soluble drugs and poorly soluble drugs, respectively. Coated with polyethylene glycol, PEG (i.e. PEGylated), liposomes have been extensively investigated because of their capability to remain sufficiently long in the blood in order to accumulate in various pathological areas(passive targeting) with the compromised leaky vasculature, such as tumors [D. D. Lasic, F. J. Martin (Eds.), Stealth Liposomes, CRC Press, Boca Raton, 1995.].
It was demonstrated in 1988 that the 86-mer trans-activating transcriptional activator, Tat, protein encoded by HIV-1, was efficiently internalized by cells in vitro when introduced in the surrounding media [M Green, P. M. Loewenstein, Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein, Cell 55 (1988) 1179-1188; A. D. Frankel, C. O. Pabo, Cellular uptake of the tat protein from human immunodeficiency virus, Cell 55 (1988) 1189-1193.]. It has been shown that short peptides could provide an active transport mechanism to facilitate nanoparticles to cross cell membranes and enter cells and nucleus [R. D. Egleton, T. P. Davis, Bioavailability and transport of peptides and peptide drugs into the brain, Peptides 18 (1997) 1431-1439.]. In this approach, which has become a useful and effective technique for overcoming the cellular barrier for intracellular drug delivery, certain proteins or peptides can be tethered to drug to form a construct that exhibits the capability to translocate across the plasma membrane and deliver the payload intracellularly. These proteins or peptides contain domains of less than 20 amino acids,are often referred to as Protein Transduction Domains (PTDs), or cell-penetrating peptides (CPPs). Nuclear Targeting Peptides (NTPs) are CPPs that allow intracellular transport of drug systems inside the nucleus.
A wide variety of peptides, either derived from proteins or synthesized chemically, have been developed and used for cellular membrane translocation. These peptides include Antennapedia (Antp) [A. Joliot, C. Pernelle, H. Deagostini-Bazin, A. Prochiantz, Antennapedia homeobox peptide regulates neural morphogenesis, Proc. Natl. Acad. Sci. USA 88 (1991) 1864-1868], VP22 [G. Elliott, P. O'Hare, Intercellular trafficking and protein delivery by a herpesvirus structural protein, Cell 88 (1997) 223-233], transportan [M. Pooga, M. Hallbrink, M. Zorko, U. Langel, Cell penetration by transportan, FASEB J. 12 (1998) 67-77.], model amphipathic peptide MAP [J. Oehlke, et al., Cellular uptake of an alpha-helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocytically, Biochim. Biophys. Acta 1414 (1998) 127-139.], signal sequence-based peptides [M. Rojas, J. P. Donahue, Z. Tan, Y. Z. Lin, Genetic engineering of proteins with cell membrane permeability, Nat. Biotechnol. 16 (1998) 370-375.1 and synthetic polyarginines [S. Futaki, et al., Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery, J. Biol. Chem. 276 (2001) 5836-5840.]
TAT peptide (TATp), which is derived from the transcriptional activator protein encoded by human immunodeficiency virus type 1 (HIV-1) [K. T. Jeang, H. Xiao, E. A. Rich, Multifaceted activities of the HIV-1transactivator of transcription, Tat, J. Biol. Chem. 274 (1999) 28837-288401]has been a widely used CPP system. The transduction ability of Tat protein is due to the positive charge in the transduction domain of TAT (TATp), which extends from residues 47-57: Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg, which contains six arginines (Arg) and two lysine residues [S. R. Schwarze, K. A. Hruska, S. F. Dowdy, Protein transduction: unrestricted delivery into all cells? Trends Cell Biol. 10 (2000) 290-295.]
Josephson et al provided the first example of CPP-mediated nanoparticulate delivery in 1999 [L. Josephson, C. H. Tung, A. Moore, R. Weissleder, High-efficiency intracellular magnetic labeling with novel superparamagnetic-Tat peptide conjugates, Bioconjug. Chem. 10 (1999) 186-191. The fluorescence microscopy studies on the live cells revealed that The conjugate was shown to accumulate first in lysosomes, followed by intense localization within the nuclei.
Other CPP systems have also been used for cellular uptake and drug delivery. MAP has the fastest uptake, followed by transportan, TATp (48-60), and penetratin. Similarly, MAP has the highest cargo delivery efficiency, followed by transportan, TATp (48-60), and penetratin. For a review, see Ref [Vladimir P. Torchilin, Tat peptide-mediated intracellular delivery of pharmaceutical nanocarriers, Advanced Drug Delivery Reviews 60 (2008) 548-558, and reference therein]